Small tyrosine kinase inhibitors interrupt EGFR signaling by interacting with erbB3 and erbB4 in glioblastoma cell lines

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available at www.sciencedirect.com

www.elsevier.com/locate/yexcr

Research Article

Small tyrosine kinase inhibitors interrupt EGFR signaling by interacting with erbB3 and erbB4 in glioblastoma cell lines Estefanía Carrasco-García a,b , Miguel Saceda a,b , Silvina Grasso a , Lourdes Rocamora-Reverte a , Mariano Conde a , Ángeles Gómez-Martínez a , Pilar García-Morales a,b , José A. Ferragut a , Isabel Martínez-Lacaci a,c,⁎ a

Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, 03202 Elche (Alicante), Spain Unidad de Investigación, Hospital General Universitario de Elche, 03203 Elche (Alicante), Spain c Unidad AECC de Investigación Traslacional en Cáncer, Hospital Universitario Virgen de la Arrixaca, 30120 Murcia, Spain b

A R T I C L E I N F O R M A T I O N

A B S T R A C T

Article Chronology:

Signaling through the epidermal growth factor receptor (EGFR) is relevant in glioblastoma. We have

Received 11 November 2010

determined the effects of the EGFR inhibitor AG1478 in glioblastoma cell lines and found that U87 and

Revised version received

LN-229 cells were very sensitive to this drug, since their proliferation diminished and underwent a

16 March 2011

marked G1 arrest. T98 cells were a little more refractory to growth inhibition and A172 cells did not

Accepted 17 March 2011

undergo a G1 arrest. This G1 arrest was associated with up-regulation of p27kip1, whose protein

Available online 1 April 2011

turnover was stabilized. EGFR autophosphorylation was blocked with AG1478 to the same extent in all the cell lines. Other small-molecule EGFR tyrosine kinase inhibitors employed in the clinic, such as

Keywords:

gefitinib, erlotinib and lapatinib, were able to abrogate proliferation of glioblastoma cell lines, which

EGFR

underwent a G1 arrest. However, the EGFR monoclonal antibody, cetuximab had no effect on cell

Receptor tyrosine kinase inhibitors

proliferation and consistently, had no effect on cell cycle either. Similarly, cetuximab did not inhibit

Cetuximab

proliferation of U87 ΔEGFR cells or primary glioblastoma cell cultures, whereas small-molecule EGFR

Cell cycle

inhibitors did. Activity of downstream signaling molecules of EGFR such as Akt and especially ERK1/2

Glioblastoma

was interrupted with EGFR tyrosine kinase inhibitors, whereas cetuximab treatment could not sustain this blockade over time. Small-molecule EGFR inhibitors were able to prevent phosphorylation of erbB3 and erbB4, whereas cetuximab only hindered EGFR phosphorylation, suggesting that EGFR tyrosine kinase inhibitors may mediate their anti-proliferative effects through other erbB family members. We can conclude that small-molecule EGFR inhibitors may be a therapeutic approach for the treatment of glioblastoma patients. © 2011 Elsevier Inc. All rights reserved.

Introduction Malignant gliomas are the most frequent cancers of the central nervous system, among which, the subtype glioblastoma multiforme

is the most common, aggressive and difficult to treat. Current therapy includes surgery, radiation and chemotherapy [1]. However, these tumors show resistance to chemotherapy and radiation. The most frequent genetic lesions found in glioblastomas are TP53

⁎ Corresponding author at: Instituto de Biología Molecular y Celular, Ed. Torregaitán, Universidad Miguel Hernández, 03202 Elche (Alicante), Spain. Fax: + 34 96 6658758. E-mail addresses: [email protected], [email protected] (I. Martínez-Lacaci). Abbreviations: BrdU, bromodeoxyuridine; Cdk, cyclin-dependent kinase; DMSO, dimethyl sulfoxide; EGFR, epidermal growth factor receptor; FBS, fetal bovine serum; RTK, receptor tyrosine kinase 0014-4827/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2011.03.015

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mutations, EGFR amplifications and PTEN mutations [2], which leads to inactivation of the p53 and retinoblastoma pathway, dysregulation of growth factor signaling and activation of the phosphatidylinositol 3-kinase (PI3K) pathway, respectively [3]. Epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase (RTK) that upon ligand binding will undergo a conformational change that will result in homo- and/or heterodimerization with other members of the family such as erbB2 (HER2/neu), erbB3 (HER3), or erbB4 (HER4) [4,5], followed by ATP binding to the catalytic site and phosphorylation in tyrosine residues in the activation loop of the catalytic domain. This results in activation of the receptor tyrosine kinase, which phosphorylates other tyrosine residues in the cytoplasmic domain that serve as docking sites for Src homology 2 (SH2) and/or phosphotyrosine binding (PTB) domains of several adaptors or effector molecules [6]. These molecules will transduce a downstream signal either through the extracellular signal-regulated kinase (ERK)/mitogen-activated kinase (MAPK) pathway or the PI3K pathway, involved in cellular processes such as proliferation and survival [6]. The erbB family represents an interactive network of transmembrane RTK and the prevalence of homo- and hetero-dimer formation follows a hierarchical order determined by the abundance and presence of not only receptors but ligands as well [4,5,7]. A link between EGFR signaling and alterations in normal cell cycle progression has been suggested in several reports. Externally, signaling pathways such as the Ras/Raf/Mek/Erk and the PI3K/Akt pathway can control cyclin concentrations. D-type cyclins and their associated kinases act as sensors of the external stimuli elicited by membrane-bound receptors and permit cells to proceed through the G1 phase of the cell cycle [8]. Cyclin D-cyclin dependent kinases (cdk) 4 and 6, cyclin E-cdk2 and cyclin A-cdk2 operate consecutively in the progression through the G1 and S phases [9]. Cdk's phosphorylate retinoblastoma (Rb), which is the limiting factor in the G1/S transition. When Rb is phosphorylated, it releases E2F factors allowing S phase progression. Cdk activity is regulated at different levels. Important cdk regulators are the INK (p15, p16, p18, and p19) and Cip/Kip (p21, p27, and p57) families of cdk inhibitors [10]. Therefore, EGFR-elicited signal transduction pathway may play a role in glioblastoma initiation and tumor progression. The use of small-molecule inhibitors and monoclonal antibodies targeted against EGFR is a common therapeutic strategy in several solid tumors. In this report, we have used the EGFR tyrosine kinase inhibitors AG1478, gefitinib (Iressa, ZD1839), erlotinib (Tarceva, OSI-774) and lapatinib (GW572016, Tykerb®), and the antibody cetuximab (Erbitux, C225) to assess their role in glioblastoma cell proliferation. We have found that AG1478, gefitinib, erlotinib and lapatinib are able to inhibit proliferation of human glioblastoma cell lines and primary cultures, whereas cetuximab has no antiproliferative effect. Furthermore, smallmolecule EGFR inhibitors induce a G1 arrest that is associated with reduction in the steady-state levels of proteins involved in the G1 checkpoint (cyclin D1, cyclin D3, cdk6, cdk4 and cdk2) and increase of cdk inhibitor protein levels (p27kip1 and p21cip1). Consistent with the lack of anti-proliferative effects, cetuximab has no effect on cell cycle, even though it is able to inhibit EGFR phosphorylation. Our data suggest that, as opposed to cetuximab, EGFR tyrosine kinase inhibitors are able to sustain inactivation of EGFR downstream molecules such as Akt and ERK1/2, and they may elicit their effects by inhibiting other members of the EGFR family such as erbB3 or erbB4, whereas cetuximab only inactivates EGFR. Taken together, this data indicate that small-molecule EGFR inhibitors may be a

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promising and powerful strategy for the treatment of glioblastoma, either as single agents or in combination with other therapeutic drugs or radiotherapy.

Materials and methods Reagents AG1478 was purchased from Calbiochem (San Diego, CA). Gefitinib, erlotinib and lapatinib were obtained from ChemieTek (Indianapolis, IN). Cetuximab was kindly donated by the Department of Pharmacology of the Hospital General Universitario de Elche (Elche, Spain). Propidium iodide, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT), and bromodeoxyuridine (BrdU) were purchased from Sigma-Aldrich (St. Louis, MO).

Cell culture The human glioblastoma cell lines U87-MG (U87), A172, T98G (T98) and LN-229 were obtained from the American Type Culture Collection (Manassas, VA, USA). The U87 ΔEGFR cell line was kindly donated by the Memorial Sloan-Kettering Cancer Center (New York, NY, USA). Primary glioblastoma cell culture samples were obtained from surgical aspirates after tumor resection performed in patients at the Hospital General Universitario de Elche (Elche, Alicante, Spain), and provided by the Hospital Biobank according to institutional human ethic guidelines. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heatinactivated fetal bovine serum (FBS), 2 mM glutamine, 1 mM sodium pyruvate, 50 U/mL of penicillin and 50 mg/mL streptomycin and incubated at 37 °C in a humidified 5% CO2/air atmosphere.

Cell proliferation assays Cells were plated in 96-well plates at a density of 2–3 × 106 cells per well, depending on the cell line, treated with vehicle (DMSO) or different doses of EGFR inhibitors using six wells per treatment for 72 h. Then, the MTT reagent was added and incubated for 3 h at 37 °C in a humidified 5% CO2/air atmosphere. After the incubation, the media were aspirated and 200 μL of DMSO were added to each well to dissolve the formazan product. After shaking for 30 min, the absorbance was measured at 570 nm in a microplate reader (Anthos 2001 Labtec Instruments GmbH, Wals, Austria).

Flow cytometry Cells were plated and treated with EGFR inhibitors for particular times. Cells were trypsinized, washed with PBS and fixed with 75% cold ethanol at −20 °C for at least 1 h. Then, cells were incubated with 0.5% Triton X-100 and 25 μg/mL RNase A in PBS, stained with 25 × 10− 3 μg/mL propidium iodide, incubated for 30 min in the dark and analyzed using an Epics XL flow cytometer (Beckman Coulter Co., Miami, FL) to determine cell cycle distribution of DNA content.

BrdU incorporation Cells were plated and treated with DMSO or AG1478 as mentioned above. Cells were pulsed with BrdU for 16 h before harvesting and then, were trypsinized, washed with PBS and fixed

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with 75% ethanol. Subsequently, DNA was denatured with 2 N HCl/0.5% Triton X-100/PBS, and the samples were blocked with PBS containing 1% bovine serum albumin (BSA) and incubated for 1 h at room temperature in the dark with an anti-BrdU antibody conjugated to FITC, or the isotypic control antibody-FITC (BD Pharmingen, San Diego, CA). Cells were washed, incubated with PBS containing 5 μg/mL propidium iodide and RNase A to counterstain DNA, and analyzed by flow cytometry using an Epics XL flow cytometer.

incubated with primary antibodies against EGFR, erbB2, erbB3, erbB4, p27kip1, p21cip1, cyclin D1, cyclin D3, cdk4, cdk6, cdk2, phospho-Akt, Akt and phospho-erbB4 and from Santa Cruz Biotechnology (Santa Cruz, CA), Rb (BD Pharmingen), phosphoerbB3, phospho-ERK1/2 and ERK1/2 from Cell Signaling Technologies (Beverly, MA), or β-actin (Sigma-Aldrich) and incubated with horseradish peroxidase-linked secondary antibodies (Amersham, GE Healthcare, Buckinghamshire, UK). Proteins were detected by the ECL system (Amersham, GE Healthcare). Densitometric analyses were performed using the Scion Image software.

Western Blot analysis

Immunoprecipitation

Cells were plated for different times, treated with EGFR inhibitors and lysed in NP-40 lysis buffer (50 mM Tris–HCl pH 7.4, 1% NP-40, 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 30 mM Na4P2O7, and 1 mM Na3VO4,) with protease inhibitor cocktail (Sigma-Aldrich) for 30 min on ice. After centrifugation at 15,000 × g for 5 min at 4 °C, the supernatants were collected and protein concentrations were determined by the Bradford method (Bio-Rad, Richmond, CA). Then, 50 μg of protein from each lysate was resolved by SDSPAGE, transferred to nitrocellulose membranes, blocked for 1 h,

A

Cells were treated, harvested and lysed as above. Then, 800– 1300 μg of protein were incubated with antibodies against EGFR or p27kip1 (Santa Cruz Biotechnology) or a control IgG (SigmaAldrich), and rotated end over end overnight at 4 °C, followed by incubation for 1 h at 4 °C with protein G-Agarose beads (Pierce Chemical Co., Rockford, IL). Beads were washed four times with lysis buffer and resuspended in SDS-PAGE sample buffer. Proteins

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Fig. 1 – Effects of AG1478 on cell proliferation and cell cycle. (A) LN-229, T98, A172, and U87 were grown in 96-well plates, treated with DMSO or different concentrations of AG1478 for 3 days, incubated with MTT for 3–4 h, dissolved with DMSO and cell proliferation rates were determined by colorimetry, represented as the average of 8–10 separate experiments and referred as percentage of control. Error bars are the S.E.M. (B) LN-229, T98, A172, and U87 were grown, treated with DMSO or 10 μM AG1478 for 24 h and cell cycle distribution was determined by flow cytometry, as described in Materials and methods. (C) Cell cycle distribution of DNA content in LN-229 cells after AG1478 10 μM treatment for 16, 24, 48 and 72 h. BrdU incorporation. (D) LN-229 cells were plated and treated with DMSO or 10 μM AG1478 for the times indicated and DNA synthesis was determined by BrdU incorporation, as described in Materials and methods. The percentages of positive cells were represented as the average of four experiments. Error bars are the S.E.M. **, significantly different from control at p < 0.01 (Student's t test).

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were eluted by boiling the beads and were subjected to Western blot analysis, using antibodies already mentioned or an antibody against phospho tyrosine (Santa Cruz Biotechnology).

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end as the reporter dye and with a non fluorescent quencher dye in the 3′ end. Relative gene expression levels of p27kip1 mRNA were calculated by the comparative Ct method referred to the GAPDH mRNA housekeeping gene expression.

Real time quantitative RT-PCR Phospho-receptor tyrosine kinase (RTK) array To determine the level of p27kip1 mRNA, total RNA from DMSOtreated or AG1478-treated cells was isolated using the RNeasy Plus kit (Qiagen). Reverse transcription of 1 μg RNA was performed using the TaqMan Reverse Transcription Reagents kit (Applied Biosystems, Foster City, CA), according to the manufacturers' instructions. Real time quantitative PCR was performed to amplify 20 ng of cDNA using the 7300 Real Time PCR System (Applied Biosystems). Levels of p27kip1 mRNA expression was determined using the Hs01597588_m1 TaqMan Gene Expression Assay (Applied Biosystems, Foster City, CA), which contains a probe that is labeled with 6-FAM in the 5′ end as the reporter dye, and with a non fluorescent quencher in the 3′ end. Expression of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was used as endogenous reference in multiplex PCR. GAPDH expression was determined using TaqMan Endogenous Control (4326317E) of Applied Biosystems, whose probe is labeled with VIC in the 5′

Cells were plated, treated with EGFR inhibitors and lysed in NP-40 lysis buffer (1% NP-40, 10% glycerol, 20 mM Tris–HCl pH 8, 137 mM NaCl, 2 mM EDTA, 1 mM sodium orthovanadate and protease inhibitors). Protein concentrations were determined by the Bradford method and 500 μg of each lysate was diluted and incubated with the Human Phospho-RTK Array (R&D Systems, Minneapolis, MN), according to the manufacturer's instructions. In brief, capture and control antibodies were spotted in duplicate on nitrocellulose membranes. Cell lysates were incubated overnight on the membranes and after binding the extracellular domain of both phosphorylated and unphosphorylated RTKs, unbound material was washed away. A pan anti-phospho-tyrosine antibody conjugated to horseradish peroxidase was then used to detect phosphorylated tyrosines on activated receptors by the ECL method.

Fig. 2 – Expression and activation of EGFR. (A) EGFR protein levels were determined by Western blot analysis in A172, U87, T98 and LN-229 cells. EGFR phosphorylation levels. (B) Cells were grown in 10% FBS-containing media (FBS) and serum-starved for 24 h. Then, cells were non-treated (control), or pre-treated with 10 μM AG1478 (AG) for 30 min and challenged with 100 ng/mL EGF for additional 10 min. EGFR was immunoprecipitated and subjected to Western blot using antibodies against p-Tyr and EGFR. A control IgG is included.

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Results AG1478 is able to inhibit proliferation of glioblastoma cell lines U87, T98, A172 and LN-229 glioblastoma cell lines were growth inhibited with AG1478 in a concentration-dependent manner (Fig. 1A). U87 and LN-229 cells were more sensitive to the drug with 82.4±2.7% and 60.8±2.4% of inhibition respectively, after 72 h of treatment with 10 μM AG1478. T98 and A172 cells were somewhat more refractory to growth inhibition and underwent 42.2 ±2.7% and 35.5 ±1.6% of inhibition respectively, under the same conditions.

Glioblastoma cell lines undergo a G1 arrest in response to AG1478 We performed cell cycle analyses and found that LN-229, U87, and to a lesser extent, T98 glioblastoma cells accumulated in the G1 phase of the cell cycle upon AG1478 treatment (Fig. 1B). LN-229 cells were especially sensitive to AG1478 treatment, and the G1 arrest was

observed as early as 16 h and maintained for 72 h (Fig. 1C). U87 cells were very sensitive to AG1478 treatment as well. However, the G1 arrest was appreciated in T98 cells only at 48 and 72 h (data not shown) and A172 cells were not arrested in the G1 phase of the cell cycle. BrdU incorporation studies confirmed that LN-229 cells treated with AG1478 did not enter into S phase (Fig. 1D).

Expression and activation of EGFR in human glioblastoma cell lines The levels of EGFR protein in U87, T98, A172 and LN-229 glioblastoma cell lines were determined by Western blot analysis (Fig. 2A). The four cell lines expressed EGFR protein, but the levels of expression were different. A172 cells express a mutant form of EGFR named TDM/18–26 that has been already described [11]. In order to assess whether AG1478 inhibit EGFR activation, we determined the EGFR phosphorylation status in serum-deprived cells in response to EGF for 10 min in the presence or absence of AG1478 (Fig. 2B). We determined that AG1478 was able to inhibit the phosphorylation of EGFR in all the cell lines, including the variant EGFR TDM/18–26 form present in A172 cells.

Fig. 3 – Effects of AG1478 on G1 checkpoint proteins. (A) LN-229 cells were left untreated (−) or treated with 10 μM AG1478 (+) for 4, 8, 16, 24, 48 and 72 h and subjected to Western blot analysis using the antibodies indicated. β-actin was used as a loading control. Rb activation analysis. (B) LN-229 cells were non-treated (−) or treated with 10 μM AG1478 (+) for 24, 48 and 72 h and subjected to Western blot analysis using an antibody against Rb.

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AG1478 effect on proteins involved in the G1 checkpoint

Role of p27kip1 on AG1478-mediated G1 arrest

In order to determine the mechanism by which AG1478 induce a G1 arrest in glioblastoma cell lines, we analyzed the steady-state protein levels of D-cyclins, cdk4, cdk6, cdk2, and the cdk inhibitors p21cip1 and p27kip1 in non-treated or AG1478-treated LN-229 cells for various times (Fig. 3A). Cyclin D1 protein levels began to diminish after 8 h of treatment. The cyclin D3 decrease was more moderate and took place at a later time, being apparent after 48 h. The protein levels of cdk4, cdk6 and cdk2 diminished after 8 h of treatment. But the most striking effect was the induction of p21cip1 and more importantly, p27kip1 protein levels that began to take place after only 4–8 h of treatment with the EGFR inhibitor AG1478. We studied the Rb activation status in response to AG1478 (Fig. 3B) and determined that Rb was hypophosphorylated in the presence of AG1478, corroborating the G1 arrest induced by this tyrosine kinase inhibitor.

In order to establish whether the G1 arrest was mediated by p27kip1, we studied the association of p27kip1 with cdk's by immunoprecipitation, followed by Western blot analyses (Fig. 4A). The association of cdk2, cdk4 and cdk6 with p27kip1 increased over time after treatment with the EGFR inhibitor in LN-229 cells. We also studied the p27kip1 protein levels after AG1478 treatment in all the glioblastoma cell lines and found that p27kip1 protein levels increased in LN-229 (Fig. 3A), U87 and T98 cells (Fig. 4B), but not in A172 cells (Fig. 4B), which do not undergo a G1 arrest, suggesting that p27kip1 is involved in the G1 arrest caused by AG1478-mediated EGFR inhibition in glioblastoma cells. In order to establish the mechanism involved in p27kip1 protein upregulation, we analyzed the p27kip1 mRNA levels in LN-229 treated cells (Fig. 4C) and found that p27kip1 mRNA induction in response to AG1478 was modest and occurred at a later time (24–48 h),

Fig. 4 – Association of p27kip1 with cdk's. (A) LN-229 cells were left untreated (−) or treated with 10 μM AG1478 (+) for 4, 8 and 24 h. Cells were harvested, p27kip1 protein was immunoprecipitated and its association with cdk2, cdk4 and cdk6 was determined by Western blot. Effects of AG1478 on p27kip1 protein steady-state levels. (B) U87, T98 and A172 cells were non-treated (C) or treated with 10 μM AG1478 (AG) for 24 h, harvested and subjected to Western blot analysis using antibodies against p27kip1 and β-actin. Effects of AG1478 on p27kip1 mRNA levels. (C) LN-229 cells were plated and non-treated or treated with 10 μM AG1478 (+) for 4, 8, 24 and 48 h, and p27kip1 mRNA levels were determined by RT-PCR, as indicated in Materials and methods. Effect of AG1478 on p27kip1 protein turnover. (D) LN-229 cells were left untreated (control) or treated (AG1478) with 10 μM AG1478 for 16 h, washed, and incubated in fresh medium containing 10 μg/mL cycloheximide (CHX). After incubation for different times, cells were harvested and p27kip1 levels were analyzed by Western blot. Protein bands were quantified, represented as the average of three separate experiments and referred as percentage of control. Error bars represent the S.E.M.

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when p27kip1 protein levels have already increased (4 h). In order to determine whether AG1478 had an effect on p27kip1 protein stability, we performed protein half-life studies in the presence of cycloheximide (CHX) and found that indeed the EGFR inhibitor was able to stabilize p27kip1 protein in LN-229 cells (Fig. 4D).

Effects of other EGFR inhibitors on cell proliferation We wanted to examine if other EGFR inhibitors apart from AG1478 were also able to inhibit proliferation of glioblastoma cell lines. We used other two small-molecule EGFR inhibitors that are being utilized in the clinic: gefitinib (Iressa) and erlotinib (Tarceva) and also cetuximab (Erbitux), a monoclonal antibody targeted against EGFR which is being used for the treatment of cancer patients as well. We performed MTT assays in U87, T98, A172 and LN-229 cells and found that both gefitinib and erlotinib were able to inhibit cell proliferation (Figs. 5A and B). LN-229 was the most sensitive cell line to erlotinib, reaching 55.1 ± 2.5% of inhibition at 10 μM, followed by U87 with 44 ± 3.6%, A172 with 37.9 ± 2.9% and T98 with 29.7 ± 2.3%. Gefitinib was somewhat less effective with percentages of inhibition ranging from 47.7 ± 3.3% in A172 cells to

Effects of EGFR inhibitors on cell cycle We wanted to study the effects of small-molecule EGFR inhibitors (AG1478, gefitinib and erlotinib) and cetuximab on cell cycle

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26.1 ± 1.4% in T98 at 10 μM. However, cetuximab had no effect on proliferation of glioblastoma cells, reaching only 10% of inhibition at the maximal dose employed (100 μg/mL) (Fig. 5C). In addition, cetuximab could not inhibit proliferation of the U87 ΔEGFR cell line, which carries the EGFRvIII mutation frequently found in gliomas [12,13] (Supplementary Fig. 1A), but small-molecule inhibitors such as AG1478 could (Supplementary Fig. 1B). Interestingly enough, lapatinib (GW572016, Tykerb®), a more specific EGFR inhibitor, was able to inhibit proliferation of LN-229, T98, A172, U87 and U87 ΔEGFR cells to a similar extent than the other small-molecule inhibitors used (Figs. 6A and B). Moreover, EGFR small-molecule inhibitors were able to inhibit the proliferation of primary cell cultures obtained from surgical aspirates from three different patients with glioblastoma, but cetuximab had no effect (Fig. 7), corroborating the results obtained with the established cell lines.

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cetuximab (µg/mL) Fig. 5 – Effects of erlotinib, gefitinib and cetuximab on cell proliferation and cell cycle. LN-229, T98, A172, and U87 were grown in 96-well plates, non-treated or treated with different concentrations of EGFR inhibitors for 72 h. Cell proliferation rates were determined by MTT assays, as described in Materials and methods, represented as the average of 6–8 separate experiments and referred as percentage of control. Error bars are the S.E.M. (A) Effects of erlotinib. (B) Effects of gefitinib. (C) Effects of cetuximab. Effects of EGFR inhibitors on cell cycle distribution. (D) LN-229 cells were grown, non-treated (control) or treated with 10 μM AG1478 (AG), 10 μM erlotinib (Erl), 10 μM gefitinib (Gef) or 50 μg/mL cetuximab (Cet) for 24 h and cell cycle distribution of DNA content was determined, as described in Materials and methods. Numbers indicate % of cells in the G1 phase.

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Fig. 6 – Effects of lapatinib on cell proliferation. Glioblastoma cell lines were grown in 96-well plates, non-treated or treated with lapatinib for 72 h. Cell proliferation rates were determined by MTT assays, as described in Materials and methods, represented as the average of separate experiments and referred as percentage of control. Error bars are the S.E.M. (A) Lapatinib dose–response effect on proliferation of LN-229 cells. (B) Effects of 0.1 and 10 μM lapatinib on proliferation of glioblastoma cell lines.

distribution. We performed cell cycle analyses after 24 h of treatment in LN-229 cells (Fig. 5D) and found that AG148, gefitinib and erlotinib caused a G1 arrest in these cells. Lapatinib caused a G1 arrest as well (data not shown). However, cetuximab had no effect on cell cycle distribution, as a result of its ineffectiveness on cell proliferation.

Study of EGFR inhibitors on activation of EGFR and downstream signaling molecules In order to assess whether small-molecule EGFR inhibitors and cetuximab had an effect on EGFR activation, we determined the EGFR phosphorylation status in serum-deprived cells in response to EGF for 10 min in the presence or absence of cetuximab (Fig. 8A), gefitinib or erlotinib (Fig. 8B) and found that indeed the three anti-EGFR agents were able to abrogate phosphorylation of EGFR wild type and mutant forms in EGF-challenged A172 cells. Next, we studied the effects of EGFR inhibitors on signaling pathways downstream of EGFR and analyzed Akt and ERK1/2 activation in LN-229 cells treated with AG1478 or cetuximab. We found that both inhibitors were able to reduce ERK1/2 and Akt

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Fig. 7 – EGFR inhibitors effects on primary glioblastoma cell cultures. HGUE-GB-1, HGUE-GB-2 and HGUE-GB-3 primary cell cultures were obtained from surgical aspirates performed during tumor resections of three different patients. Cells were grown in 96-well plates, non-treated or treated with different EGFR inhibitors for 72 h. Cell proliferation rates were determined by MTT assays, as described in Materials and methods, represented as the average of three separate experiments and referred as percentage of control. Error bars are the S.E.M. HGUE-GB-3 is presented as a representative sample.

phosphorylation levels after 4 h of treatment (Fig. 8C). However, this reduction was maintained over time only with AG1478 but not with cetuximab, indicating that cetuximab was not able to hinder these pathways after a long period of time (48 h). This could explain the lack of effects of cetuximab on cell cycle and cell proliferation. We also found that the inhibition of ERK1/2 phosphorylation was completely abrogated after long periods of treatment with both gefitinib and erlotinib, but not the phosphorylation of Akt (Fig. 8D), which indicates that ERK1/2 seems to be the common effector of small-molecule inhibitors and it is more important than Akt.

Role of other receptor tyrosine kinases (RTK) We wanted to determine whether EGFR inhibitors affect the activity of other tyrosine kinase receptors. A phospho-RTK array in non-treated (control) and AG1478-treated LN229 cells (Fig. 9A) revealed that not only levels of phosphorylated EGFR but also of erbB3 and to a lesser extent of erbB2 and erbB4 phosphorylation diminished after 30 min of treatment. The reduction of EGFR respective to control conditions was 41.9% and the reduction of erbB3 was 54.8%. The control RTK array corresponds to cells grown in the presence of serum that is able to stimulate EGFR and erbB3 in LN-229 cells. Other RTK that appeared stimulated under these conditions were InsR, IGF-1R and PDGFβR. The phosphorylation of these receptors was unaffected by AG1478 treatment. However, in serum-starved LN-229 cells EGFR, erbB3, Axl, Dtk and Eph receptors appeared phosphorylated, indicating that these receptors may be constitutively activated. The stimulation of EGFR in serum-starved LN-229 cells can also be appreciated in Fig. 2B.

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Role of other erB receptors Since erbB3 phosphorylation was strongly affected by AG1478 in LN-229 cells, we set out to analyze the protein levels of erbB2, erbB3 and erbB4 in glioblastoma cell lines to evaluate their potential as targets of EGFR inhibitors (Fig. 9B) and found that erbB2 protein levels were low in U87 and A172 cells, a little bit higher in LN-229 cells, and barely detectable in T98 cells. However, LN-229 cells express high levels of erbB3 and the four cell lines express erbB4, especially U87 and LN-229 cells. As observed in Figs. 9C and D, erbB3 phosphorylation levels diminished after treatment with the four small-molecule EGFR inhibitors (AG1478, erlotinib, gefitinib and lapatinib), corroborating the results obtained with the RTK arrays (Fig. 9A), but not with cetuximab. In addition, ErbB4 phosphorylation levels decreased mainly after AG1478 and erlotinib treatment, slightly less after gefitinib and lapatinib treatment, but cetuximab had no effect at all (Figs. 9C and D). These findings suggest that small tyrosine kinase inhibitors may mediate their effects through other erbB receptors apart from EGFR. However, cetuximab is able to inhibit phosphorylation of only EGFR, and this is the basis explaining the differential effects between the small-molecule EGFR inhibitors and cetuximab.

Discussion Glioblastoma is very difficult to treat and an aggressive type of cancer characterized by poor clinical outcome. In this report, we have shown that small-molecule tyrosine kinase inhibitors specific for EGFR are able to inhibit glioblastoma cell proliferation by causing a G1 arrest in the cell cycle. The tyrosine phosphorylation inhibitor 4(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) is a specific and very potent inhibitor of the EGFR tyrosine kinase that has shown promising preclinical results [14]. In fact, AG1478 was able to inhibit EGFR activity in a dose-dependent manner in mice bearing xenografts of the U87MG.Δ2-7 (or ΔEGFR) cell line, which overexpresses the constitutive active mutant EGFR variant EGFRvIII [15]. It has been shown to have anti-proliferative effects in a variety of tumor cell lines [16–18] and to augment the sensitivity to cytotoxic drugs like cisplatin and doxorubicin [19]. AG1478 shares the same structural quinazoline backbone that the clinically used drugs gefitinib and erlotinib, but lacks the hydrophilic side chains that may confer different properties to gefitinib and erlotinib. These molecules are competitive reversible inhibitors of the ATP binding site in the kinase domain. We have shown that AG1478 inhibits

Fig. 8 – Effects of EGFR inhibitors on EGFR and downstream effectors. Cetuximab effects on EGFR phosphorylation. (A) A172 cells were grown in 10% FBS-containing media (FBS) and serum-starved for 24 h. Then, cells were non-treated (control), or pre-treated with 50 μg/mL cetuximab (Cet) for 30 min and challenged with 100 ng/mL EGF for additional 10 min. EGFR was immunoprecipitated and subjected to Western blot using antibodies against p-Tyr and EGFR. A control IgG is included. Erlotinib and gefitinib effects on EGFR phosphorylattion. (B) Serum-starved A172 cells were treated with 10 μM erlotinib (Erl) or 10 μM gefitinib (Gef) and 100 ng/mL EGF and subjected to inmunoprecipation and Western blot. EGFR inhibitor effects on downstream molecules. (C) LN-229 cells were grown, non-treated (C) or treated with 10 μM AG1478 (AG), or 50 μg/mL cetuximab (Cet) for 4 and 48 h and levels of p-Akt, Akt, p-ERK1/2 and ERK1/2 were analyzed by Western blot. (D) LN-229 cells were grown, non-treated (C) or treated with 10 μM AG1478 (AG), 50 μg/mL cetuximab (Cet), 10 μM erlotinib (Erl), or 10 μM gefitinib (Gef) for 48 h and levels of p-Akt, Akt, p-ERK1/2 and ERK1/2 were analyzed by Western blot.

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Fig. 9 – AG1478 effects on phospho-RTK arrays. (A) LN-229 cells were grown, serum-starved for 24 h (no FBS) and replaced with media containing 10% FBS and non-treated (control) or treated with 10 μM AG1478 for 30 min (AG1478). Cell extracts were subjected to phospho-RTK, as described in Materials and methods. Inset represents erbB family receptors: EGFR (1), erbB2 (2), erbB3 (3), and erbB4 (4). Other relevant RTK are shown. Expression of erbB2, erbB3 and erbB4. (B) Protein levels of erbB2, erbB3 and erbB4 were measured by Western blot analysis in A172, U87, T98 and LN-229 cells. EGFR inhibitors effects on erbB3 and erbB4 phosphorylation. (C) LN-229 and U87 cells were plated, serum-starved for 24 h, replaced with media containing 10% FBS and non-treated (C) or treated with 10 μM AG1478 (AG), 50 μg/mL cetuximab (Cet), 10 μM erlotinib (Erl), or 10 μM gefitinib (Gef) for 30 min. p-erbB3, erbB3, p-erbB4 and erbB4 levels were analyzed by Western blot. Lapatinib effects on erbB3 and erbB4 phosphorylation. (D) LN-229 cells were plated, serum-starved for 24 h, replaced with media containing 10% FBS and non-treated (C) or treated with 10 μM Lapatinib (Lap) for 30 min. p-erbB3, erbB3, p-erbB4 and erbB4 levels were analyzed by Western blot, as described in Materials and methods.

proliferation of human glioblastoma cell lines (Fig. 1A and Supplementary Fig. 1B). The G1 arrest was more pronounced in LN-229 and U87 cells (Figs. 1B and C) and further confirmed with BrdU studies, showing that LN-229 cells did not enter S phase after AG1478 treatment (Fig. 1D). However, the different EGFR protein levels among the four cell lines and the presence of the variant EGFR form in A172 cells (Fig. 2A) or the EGFRvIII truncated form in U87 ΔEGFR cells (Supplementary Fig. 1A) cannot explain the differences in sensitivity towards AG1478 in cell proliferation or G1 arrest, as AG1478 was able to block EGFR phosphorylation after stimulation with EGF to the same extent in the four cell lines (Fig. 2B) and in the U87 ΔEGFR cell line as well [20]. It is interesting to note that the variant EGFR TDM/18–26 form present in A172 is also inhibited by the clinically utilized inhibitors, erlotinib and gefitinib (Fig. 8B). Consistent with the G1 arrest observed in LN-229 cells in response to AG1478, proteins involved in the G1 checkpoint such as D-cyclins, cdk4, cdk6 and cdk2 were downregulated. Concomitantly, the cdk

regulators p21cip1 and p27kip1 were upregulated (Fig. 3A) and Rb underwent hypophosphorylation (Fig. 3B). Since the increase in the steady-state levels of p27kip1 protein was the most notorious change observed, we further investigated whether the G1 arrest caused by AG1478 was mediated by p27kip1. We found that the association of p27kip1 with cdk4, cdk6 and cdk2 proteins increased after AG1478 treatment in LN-229 cells (Fig. 4A) and that p27kip1 protein levels augmented only in the cell lines that underwent a G1 arrest (Figs. 3A and 4B), suggesting that p27kip1 may mediate this G1 arrest. We further characterized the mechanism of p27kip1 up-regulation and found that p27kip1 mRNA levels augmented after the increase observed in the protein steady-state levels (Fig. 4C) and that p27kip1 protein turnover was stabilized after AG1478 treatment (Fig. 4D), revealing two mechanisms of up-regulation: a transcriptional mechanism and a post-transcriptional one, which results in accumulation of p27kip1 protein after AG1478 treatment. Protein half-life studies revealed that p27kip1 protein was not stabilized after

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AG1478 treatment in A172, the cell line that does not show p27kip1 accumulation (data not shown), confirming the role of p27kip1 mediating the G1 arrest due to AG1478. The small-molecule inhibitors erlotinib and gefitinib have been utilized for the treatment of advanced or metastatic non-small cell lung cancer in several phase II and III clinical trials and [21], and especially gefitinib has shown to be effective in patients carrying activating EGFR mutations [22]. Erlotinib in combination with gemcitabine has been used for the treatment of advanced pancreatic cancer in a phase III clinical trial [23]. The monoclonal antibodies cetuximab and panitumumab have been approved for the treatment of metastatic colorectal carcinoma [24] and cetuximab for head and neck squamous cell carcinoma [25]. In addition, small tyrosine kinase inhibitors (gefitinib and erlotinib) and monoclonal antibodies (cetuximab and panitumumab) have entered numerous clinical trials for the treatment of various solid tumors that depend on the EGFR signaling pathway for proliferation, including glioblastoma [26]. Similarly and more recently, the new inhibitor GW572016 (lapatinib), has entered clinical trials in patients with recurrent glioblastoma multiforme [27,28] or other types of cancer. Lapatinib inhibits EGFR and erbB2 autophosphorylation and proliferation of cells that overexpress either EGFR or erbB2 [29]. The quinazoline-based molecules AG1478, gefitinib and erlotinb bind to an active conformation of the kinase [30,31], whereas lapatinib binds to a closed conformation of the molecule and has a very slow-off rate. This slow dissociation rate may increase the duration of its effects [32]. In order to extend our studies to these clinically used EGFR inhibitors, we assessed the effect of gefitinib, erlotinib, lapatinib and cetuximab on glioblastoma cell proliferation and found that similar to AG1478, gefitinib, erlotinib and lapatinib were able to inhibit glioblastoma cell proliferation, whereas cetuximab, which interacts with the extracellular ligand binding domain, had no effect (Figs. 5A–C and 6). The cell lines utilized in this study represent various EGFR protein levels. Furthermore, the cell line A172 that harbors the EGFR TDM/18–26 form and the U87 ΔEGFR that harbors the truncated EGFRvIII form behave similarly to the other cell lines in terms of sensitivity to small-molecule inhibitors and resistance to cetuximab. Therefore, a common mechanism of the four smallmolecule EGFR inhibitors seems to take place, as opposed to cetuximab. We were interested in studying this common mechanism, and found that indeed like AG1478, gefitinib, erlotinib and lapatinib were able to induce a G1 arrest in glioblastoma cell lines, whereas cetuximab was not (Fig. 5D and data not shown). Even though smallmolecule inhibitors and cetuximab were able to reduce phosphorylation of EGFR and downstream signaling molecules such as ERK1/2 and Akt after a short treatment, the inhibition of Akt and ERK1/2 could not be sustained after long treatments with cetuximab (Figs. 8C and D), explaining its ineffectiveness on cell cycle and cell proliferation. In fact, it has been shown that gefitinib or erlotinib treatment, but not cetuximab, can further abrogate the activation of EGFR downstream signal transducers, including ERK1/2 or Akt in cetuximab-resistant cells [33]. In our system, however, ERK1/2 activity was completely abrogated after long treatments with small-molecule inhibitors, being Akt inhibition restricted to AG1478 action (Fig. 8D), which suggests that ERK1/2 is the common transducer inactivated by the tyrosine kinase inhibitors and more important than Akt. Therefore, the Ras/Raf/ Mek/Erk pathway mediates the inhibitory effects due to EGFR inhibition more than the PI3K/Akt/mTOR pathway in glioblastoma cell lines. In order to assess the specificity of the EGFR inhibitors, we employed RTK arrays and Western blot analyses and found that smallmolecule inhibitors (Figs. 2B and 8B) and cetuximab (Fig. 8A) inhibit

EGFR phosphorylation. It has been suggested that cetuximabresistance may be due to c-MET/HGF-R activation [34] and that resistance to anti-EGFR therapy is mediated by IGF-1R activation [35]. However, we have been unable to detect phosphorylation of cMET/HGF-R on RTK arrays of glioblastoma cell lines under any condition, and the stimulation of Ins R and IGF-1R by serum was not affected by EGFR inhibitors (Fig. 9A and data not shown). Interestingly enough, small-molecule inhibitors but not cetuximab were able to impede phosphorylation of other erbB receptors such as erbB3 and erbB4 (Figs. 9A, C and D), but they had no effect on other membranebound tyrosine kinases. Consistent with these results, it has been shown that AG1478 and gefitinib decreased EGFR and erbB3 phosphorylation through the inhibition of EGFR/erbB3 dimerization [36] and that erbB3 expression and heterodimerization with EGFR may influence sensitivity to erlotinib [37]. It has also been shown that AG1478 inactivates EGFR and erbB4 downstream signaling [38]. It is important to note that erbB3 lacks tyrosine kinase activity, but upon ligand stimulation can heterodimerize with other members of the family and transduce as signal, acting as a modulator [39,40]. Both EGFR and erbB4 have both ligands and robust kinase activity [41,42] and their expression levels in the glioblastoma cell lines utilized in this report are moderate to high (Fig. 9B). We can presume then that erbB3 may mediate its effects mainly by heterodimerizing with EGFR or erbB4 in this system. It has been suggested that the presence of nuclear EGFR, which is induced by EGFR ligands, contributes to resistance to cetuximab. Furthermore, its phosphorylation is inhibited by the src inhibitor dasatinib but not by cetuximab [43]. On the other hand, EGFR tyrosine kinase inhibitors (AG1478, gefitinib and erlotinib) impede EGF-induced EGFR nuclear translocation but stimulate cetuximabinduced EGFR translocation to the nucleus [44]. We have not evaluated the presence of EGFR ligands or nuclear EGFR in our system, which indeed could explain at least partially, the differential effects observed. Nevertheless, the data presented herein suggest that EGFR tyrosine kinase inhibitors are potential therapeutic drugs for the treatment of glioblastoma patients. In fact, gefitinib and erlotinib are undergoing various clinical trials in patients with malignant gliomas [26] in monotherapy or in combination with other drugs [45]. Both gefitinib and erlotinib have been generally well tolerated in phase I and phase II trials and have shown partial response [46]. Moreover, a phase II study of erlotinib plus temozolomide with radiation therapy has shown that patients receiving both drugs had better survival [47]. The beneficial effects of gefitinib in patients with high-grade glioma [46], however, are controversial, as this inhibitor seems to have antiproliferative effects only in tumors with EGFR amplification [48]. A different scenario takes place with therapies based on monoclonal antibodies, as they provoke an immunologic response. It has been shown that cetuximab drives natural killer cells towards EGFR expressing tumor cells, causing antibody-dependent cellular citotoxicity and production of immune modulatory cytokines [49,50]. Some response has been found with cetuximab on glioblastoma tumors exhibiting EGFR amplification or expressing the EGRvIII variant [51] and phase II and phase III clinical trials combining radiotherapy, temozolomide and monoclonal antibodies such as cetuximab and nimotuzumab are ongoing [46]. However, we have shown in this report that cetuximab may not be an effective therapy for the treatment of glioblastoma because interruption of the EGF signaling pathway cannot be maintained overtime and therefore, cell proliferation cannot be abrogated. Small tyrosine kinase inhibitors are more effective as they disrupt the signaling of EGFR and other members of the family such as erbB3 and erbB4 that can form heterodimers with EGFR. To further

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support our conclusions we have treated primary cell cultures obtained from patients with glioblastoma and found that indeed small-molecule EGFR inhibitors, especially AG1478 and lapatinib, reduced the proliferation of these cell cultures, whereas cetuximab did not (Fig. 7). It is important to characterize patients not only measuring EGFR status (gene amplification, presence of mutations or expression levels) but also monitoring erbB2, erbB3 and erbB4 levels, since all these receptors interact among themselves and can induce a response. A histochemical study performed in 21 human glioblastoma tumors found EGFR expression in 62% of the tumors among which no expression of the mutant EGFR variant ΔEGFR was found, 43% expressed erbB2, 95% expressed erbB3 and all of them expressed erbB4 [52], supporting the relevance of analyzing the levels of the four erbB family members in clinical studies. This may explain in part, the lack of correlation between response and EGFR amplification observed in clinical trials using gefitinib or erlotinib, and consequently, the controversial prognostic value of EGFR in glioblastoma [53]. An interesting report that reviews systematically the data obtained from 60 different clinical studies has questioned the efficacy of smallmolecule kinase inhibitors in glioblastoma [54]. The main targets that were evaluated in these studies were: EGFR, mTOR, KDR, FLT1, PKCβ and PDGFR. According to the results presented herein, these targets are not sufficient and in order to assess the effectiveness of EGFR inhibitors as therapeutic agents as it is necessary to evaluate as well erbB2, erbB3, erbB4 and ERK1/2 activity. Moreover, this compelling observation can be extended to all types of cancers treated with EGFR inhibitors.

Conclusions Our study shows that interrupting EGFR signaling in glioblastoma cell lines by using small-molecule tyrosine kinase inhibitors such as AG1478, gefitinib, erlotinib or lapatinib may be more effective than using monoclonal antibodies such as cetuximab, since the blockade of EGFR downstream molecules due to cetuximab cannot be sustained after long periods of time. Interestingly, smallmolecule inhibitors may hamper the Mek/Erk pathway more importantly than the Akt pathway. These interruptions result in reduced cell proliferation concomitant to a G1 arrest of the cell cycle that, in the case of AG1478, is associated with up-regulation of p27kip1. As opposed to cetuximab that only hinders EGFR, smallmolecule inhibitors can elicit their effects by inhibiting other members of the EGFR family such as erbB3 and erbB4. Therefore, these inhibitors are potential therapeutic drugs for the treatment of glioblastoma. Supplementary materials related to this article can be found online at doi:10.1016/j.yexcr.2011.03.015.

Acknowledgments We are especially grateful to Inmaculada Jiménez-Pulido from the Department of Pharmacology of the Hospital General Universitario de Elche (Elche, Spain), for assisting in dosage and preparation of the drugs utilized. We are also grateful to our laboratory members for helpful comments. This article has been funded by Instituto de Salud Carlos III grant FIS PI041084 to I. Martínez-Lacaci, by a grant from the Fundación de Investigación Médica Mutua Madrileña to I. Martínez-Lacaci and by Conselleria de Sanidad GVA grant AP-089/ 10 to M. Saceda.

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